biology Article Assessment of 16S rRNA Gene-Based Phylogenetic Diversity of Archaeal Communities in Halite-Crystal Salts Processed from Natural Saharan Saline Systems of Southern Tunisia Afef Najjari 1,*, Panagiota Stathopoulou 2 , Khaled Elmnasri 3, Faten Hasnaoui 1, Ines Zidi 1, Haitham Sghaier 3,4, Hadda Imene Ouzari 1, Ameur Cherif 3 and George Tsiamis 2 1 Faculté des Sciences de Tunis, LR03ES03 Laboratoire de Microbiologie et Biomolécules Actives, Université Tunis El Manar, 2092 Tunis, Tunisia; [email protected] (F.H.); [email protected] (I.Z.); [email protected] (H.I.O.) 2 Department of Environmental Engineering, Laboratory of Systems Microbiology and Applied Genomics, University of Patras, 2 Seferi Str., 30100 Agrinio, Greece; [email protected] (P.S.); [email protected] (G.T.) 3 Higher Institute for Biotechnology, University Manouba, BVBGR-LR11ES31, Biotechpole Sidi Thabet, 2020 Ariana, Tunisia; [email protected] (K.E.); [email protected] (H.S.); [email protected] (A.C.) 4 Laboratory “Energy and Matter for Development of Nuclear Sciences” (LR16CNSTN02), National Center for Nuclear Sciences and Technology (CNSTN), 2020 Sidi Thabet, Tunisia * Correspondence: [email protected] Citation: Najjari, A.; Stathopoulou, Simple Summary: In the current investigation, we determine the archaeal diversity from halite- P.; Elmnasri, K.; Hasnaoui, F.; Zidi, I.; crystal salts sampled from Chott Djerid and Chott Douz, two saline Chotts in the Tunisian Sahara, Sghaier, H.; Ouzari, H.I.; Cherif, A.; using both culture-dependent and independent approaches citing DGGE (V3 regions), 16S rRNA- Tsiamis, G. Assessment of 16S rRNA gene (full length gene ~ 1500 bp) based clone libraries and high-throughput sequencing technology of Gene-Based Phylogenetic Diversity of Illumina MiSeq platform (V3–V4 regions). The amalgamation of all results yielded a comprehensive Archaeal Communities in view of the archaeal diversity represented by members of Halobacteria and Nanohaloarchaea classes. Halite-Crystal Salts Processed from Natural Saharan Saline Systems of Abstract: A thorough assessment of the phylogenetic diversity and community structure of halophilic Southern Tunisia. Biology 2021, 10, archaea from three halite-crystal salts, processed from two separated saline systems of Southern 397. https://doi.org/10.3390/ Tunisia has been performed using culture dependent and independent methods targeting different biology10050397 regions of 16S rRNA gene sequences including DGGE, 16S rRNA clone libraries and Illumina Miseq Academic Editor: Nils-Kåre Birkeland sequencing. Two samples, CDR (red halite-crystal salts) and CDW (white halite-crystal salts), were collected from Chott-Eljerid and one sample CDZ (white halite-crystal salts) from Chott Douz. Received: 4 March 2021 Fourteen isolates were identified as Halorubrum, Haloferax, Haloarcula, and Halogeometricum genera Accepted: 30 March 2021 members. Culture-independent approach revealed a high diversity of archaeal members present in Published: 4 May 2021 all samples, represented by the Euryarchaeal phylum and the dominance of the Halobacteria class. Nanohaloarchaea were also identified only in white halite samples based on metagenomic analysis. In Publisher’s Note: MDPI stays neutral fact, a total of 61 genera were identified with members of the Halorhabdus, Halonotius, Halorubrum, with regard to jurisdictional claims in Haloarcula, and unclassified. Halobacteriaceae were shared among all samples. Unexpected diversity published maps and institutional affil- profiles between samples was observed where the red halite crust sample was considered as the most iations. diverse one. The highest diversity was observed with Miseq approach, nevertheless, some genera were detected only with 16S rRNA clone libraries and cultured approaches. Keywords: archaea; culture approach; DGGE; metagenomic; 16S rRNA gene libraries Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and 1. Introduction conditions of the Creative Commons Attribution (CC BY) license (https:// Hyper-saline lakes are widespread around the world and occur mainly in warm and creativecommons.org/licenses/by/ arid areas where rainwater has been collected, evaporated, and left large deposits of salts 4.0/). (halite, gypsum) and water with a high salt concentration [1]. They are devoid of any kinds Biology 2021, 10, 397. https://doi.org/10.3390/biology10050397 https://www.mdpi.com/journal/biology Biology 2021, 10, 397 2 of 25 of vegetation except some clumps of grass. These areas have been the subject of many microbiological studies for several years and it has been demonstrated that the diversity of halophilic and halotolerant species in such environments is limited, including taxa representing all three domains of life (Archaea, Bacteria, Eukarya) [2–9]. However, the most frequent of these microorganisms are members of the third domain of life, Archaea, especially classified within the family Halobacteriaceae, order Halobacteriales [10]. They are the main dominant microbial population, in saline systems where NaCl concentration varies from 20% (w/v) to halite saturation [8,9,11–17]. These microorganisms (Haloarchaea) have been isolated from a wide range of hypersaline ecosystems such as the Dead Sea, the Great Salt Lake (Magna, UT, USA), alkaline brines of Wadi Ntarun (west of the Nile Delta, Egypt), Solar saltern of Sfax Sabkhas (Sfax, Tunisia), Algerian sabkhas (Algeria), Greek solar saltern (Messolonghi, Greece), lake Magadi (Kenya), and saline soils [2,4,7,18–24]. Haloarchaea are known to promote the crystal halite formation [25]. In more detail, when NaCl begins to precipitate, Haloarchaea are embedded in situ within fluid inclusions and remain viable for thousands to hundreds of millions of years [26–30]. Therefore, Haloarchaea are considered as attractive models for astrobiologists due to their polyextremophilic nature with tolerance of saturating salinity, anaerobic conditions, high levels of ultraviolet and ionizing radiation, subzero temperatures, desiccation, and toxic ions [16,31,32]. Members of Haloarchaea lead to an aerobic chemo-organotrophic lifestyle and consti- tute a physiologically and morphologically distinct group. They are strongly related to high salt concentrations and require a minimum of 1.5 M NaCl concentration for maintaining growth and structural integrity of their cells [4,6,10,25,33,34]. The majority of the Haloar- chaea—such as Halobacterium, Halorubrum, Haloarcula, Haloferax, Halococcus, Halobaculum, and Natrialba spp.—exhibit an optimal growth at near neutral pH [20,35–37]. Alkaline saline environments harbor alkaliphilic halobacteria, such as Natronomonas and Natronobac- terium spp., that require at least a pH 8.5 for growth [10,38]. Within Halobacteriales at least 51 currently genera have been validly characterized, reflecting their considerable ecophysiological diversity [39,40]. During the last years research has been focused on the study of prokaryotic diversity and the structure of communities in different saline ecosystems from different geograph- ical locations, based on the analyses of PCR-amplified 16S rRNA gene sequencing from environmental samples and using techniques such as DGGE (Denaturing Gradient Gel Elec- trophoresis), 16S rRNA libraries, SSCP (Single Strand Confirmation Polymorphism), Phy- loChip, and 16S rRNA amplicon sequencing [7,20,21,41–43]. These approaches have been proved to be powerful tools to assess the microbial diversity since they enable the charac- terization of novel bacterial and archaeal lineages in such environments [6,7,9,21]. Previous studies on the microbiology of saline environments, indicated that Haloarchaea constitute the main dominant microbial group but most of them remain uncultured [21,44,45]. A differen- tiation of Haloarchaea within the different ecosystem studies was also observed [42,45–48]. Southern Tunisia includes numerous ephemeral inland and costal Saharan salt lakes, known as Sebkhas and Chotts, lying south of the Atlas Mountains at the northern edge of the Sahara [1,2]. These salt ecosystems have been the subject of microbiological studies carried on saline water, sediments, and soils [3–6]. However, to the best of our knowledge, no studies have unraveled the phylogenetic diversity of archaeal communities of halite- crystal salts from Tunisian ecosystems based on culture dependent and culture independent approaches simultaneously. The purpose of this study was to assess the archaeal diversity from halite-crystal salts sampled from Chott Djerid and Chott Douz; two saline Chotts in the Tunisian Sahara, using culture dependent and molecular approaches such as DGGE, 16S rRNA-gene based clone libraries and high-throughput sequencing technology of Illumina MiSeq platform. The present study will provide insights of the archaeal diversity from the Tunisian saline ecosystems and may be the first step towards the biotechnological exploitation of the Tunisian archaeal diversity. Biology 2021, 10, x FOR PEER REVIEW 3 of 25 hara, using culture dependent and molecular approaches such as DGGE, 16S rRNA-gene based clone libraries and high-throughput sequencing technology of Illumina MiSeq platform. The present study will provide insights of the archaeal diversity from the Tu- nisian saline ecosystems and may be the first step towards the biotechnological exploita- tion of the Tunisian archaeal diversity. Biology 2021, 10, 397 3 of 25